Project Summary Chromosomal rearrangements involving the ALK, RET, ROS and NTRK1 proto-oncogenes collectivly account approximatly 10% of molecular drivers of lung adenocarcinoma. These rearrangements result in the expression of a constitutively active fusion kinase that drives growth and proliferation. Inhibition of ALK, ROS1 and NTRK with targeted inhibitors has been very successful; however, despite the success of small molecule inhibitors in other fusion kinase driven lung cancers, patients with RET rearrangements have had very low response rates to RET inhibitors. In particular, patients that have KIF5B-RET rearrangements respond poorly compared to patients with other 5? partners such as CCDC6 or NCOA. Here, we seek to understand the role of RET 5? fusion partners in regulating response to RET inhibitors. We hypothesize that RET fusion proteins have unique molecular features, including enhanced activation of the PI3K pathway and differential usage of RET isoforms, that are dependent on the 5? fusion partner. In Aim 1, we will use two different in vivo models to determine RET inhibitor response rates in Kif5b-Ret and non-Kif5b-Ret. First, we will generate Kif5b-Ret and Trim24-Ret rearrangements with CRISPR/Cas9 technology. Second, we will use a patient-derived KIF5B-RET+ cell line and the CCDC6- RET+ LC-2/Ad cell line to create orthotopic models where we will assess RET inhibitor responses in cells expressing the human RET protein. RET is expressed as two major isoforms, RET9 and RET51. Previous studies have shown that RET51 more significantly contributes to oncogenic phenotypes and activation of the PI3K pathway than RET9. We have shown that inhibiting the PI3K pathway, with the mTOR inhibitor everolimus, can enhance sensitivity to RET inhibitors in vitro. In Aim 2, we will test the hypothesis that RET51 most significantly contributes to regulating downstream signaling as well as activation of the PI3K pathway in RET rearranged cells. We will then proceed to determine if the kinesin motor domain, contained uniquely within KIF5B- RET rearrangements, regulates RET activation and downstream pathways. This work is expected to be significant as it will reveal the molecular mechanisms that are responsible for preventing response to RET inhibitors. These results will lead to the development of predictive biomarkers of response to RET inhibitors and novel treatment strategies for patients with KIF5B-RET rearrangements.